Sensitive High-performance Liquid Chromatographic Method Research Articles

Individualized medication of venetoclax based on therapeutic drug monitoring in Chinese acute myeloid leukemia patients using an HPLC method.

The aim of this study was to establish a simple and sensitive high-performance liquid chromatography method for therapeutic drug monitoring of venetoclax (VEN) and optimize regimens. The analysis required the extraction of a 50 μl plasma sample and the precipitation of proteins using acetonitrile extraction. The chromatographic method employed a mobile phase of acetonitrile: 0.5% KH 2 PO 4 (pH 3.5) (60/40, v/v) on a Diamond C 18 (4.6 mm × 250 mm, 5 μm) column at a flow rate of 1.0 ml/min. The quantitative method was validated based on standards described in 'Bioanalytical Method Validation: Guidance for Industry' published by the US Food and Drug Administration (FDA). The calibration curve was linear ( R2 = 0.9998) over the range of 75-4800 ng/ml, with limits of quantification of 25 ng/ml. The coefficients of intraday and interday validation, specificity, recovery, and stability all met the criteria of FDA guidance. The method was successfully applied to analyze VEN concentrations in 30 cases of acute myeloid leukemia patients. The peak concentration ( Cmax ) was 1881.19 ± 756.61 ng/ml, while the trough concentration ( Cmin ) was 1212.69 ± 767.92 ng/ml in acute myeloid leukemia patients. Our study establishes a simple, precise, and sensitive high-performance liquid chromatography method for monitoring VEN and confirms its applicability for therapeutic drug monitoring of VEN in hematological cancers.

RP-HPLC-PDA-Based Simultaneous Estimation of Ramipril and Hydrochlorothiazide in Combined Dosage Forms: A Validated Approach

A rapid, highly sensitive High-Performance Liquid Chromatographic method has been developed for the simultaneous determination of Ramipril (RMP) and Hydrochlorothiazide (HCT) in bulk drug and in tablets. Ramipril (RMP) and Hydrochlorothiazide (HCT) were eluted from an Eclipse plus C18 (250 mm × 4.6 mm I.D.) with particle size 5 µm reversed phase column with a mobile phase consisting of acetonitrile and potassium dihydrogen ortho-phosphate pH 3 (40: 60 v/v) at a flow rate of 1 mL/min with UV detection at 210 nm. The retention time for RMP and HCT was found to be 3.31 ± 0.02 min and 6.64 ± 0.02 min, respectively. The linear response (r2 <0.99) was observed in the range of 2 - 12 μg/mL for RMP and 4 - 24 μg/mL of HCT with low values of limits of detection (LOD) and quantification (LOQ) for both drugs. The method shows good recoveries and intra and inter-day relative standard deviations were less than 1.0%. Validation parameters as specificity, accuracy, ruggedness and robustness were also determined. The proposed method provides accurate and precise quality control tool for routine simultaneous analysis of Ramipril and Hydrochlorothiazide in bulk and in tablet dosage form.

A simple and sensitive high-performance liquid chromatographic method combined with fluorescence detection for bioanalysis of scopoletin in rat plasma: Application to a pharmacokinetic study.

Scopoletin, a coumarin class natural phytoalexin, is present in medicinal plants such as noni (Morinda citrifolia). It exhibits diverse pharmacological properties, including antioxidant, anti-hyperuricemic, and anti-inflammatory effects. The objective of this study was to develop a novel HPLC-fluorescence (HPLC-FL) method for the quantitative analysis of scopoletin in the plasma and to investigate its pharmacokinetics in rats. Sample preparation involved a methanol-based protein precipitation method, and chromatographic separation was conducted using a C18 column with an isocratic mobile phase composed of water and acetonitrile containing 0.1% trifluoroacetic acid. The eluent was detected using an FL detector set to optimized excitation/emission wavelengths of 337/453 nm. Method validation encompassed assessments of selectivity, linearity (1-500 ng/mL), precision, accuracy, recovery, matrix effect, and stability in accordance with the prevailing Food and Drug Administration (FDA) guidelines. The developed method was successfully applied for pharmacokineticstudy in rats. To the best of our knowledge, this study is the first application of a simple and sensitive HPLC-FL method for the quantification of scopoletin in a pharmacokinetic study. This method offers a promising alternative for preclinical pharmacokinetic investigations with appropriate modifications and validations and holds potential for clinical applications.

Single Method for Determination of Biocides Benzalkonium Chloride, Chloroxylenol and Chlorhexidine Gluconate in Cosmetics, Hygiene and Health Care Products by Using High Performance Liquid Chromatography.

Benzalkonium chloride (BKC), chloroxylenol (PCMX) and chlorhexidine gluconate (CHG) are widely recognized biocidal compounds that require precise control and monitoring in various applications, including cosmetics, hygiene, health and ophthalmic products. In this study, we present a straightforward and highly sensitive high-performance liquid chromatographic (HPLC) method for the detection of these substances. The HPLC method we developed allows for the detection of BKC at concentrations as low as 1.0ppm, PCMX at 1.0ppm and CHG at 2.0ppm, all with a signal-to-noise (S/N) ratio exceeding 3.0. Additionally, the method enables quantification up to 2.0ppm for BKC, 2.0ppm for PCMX and 5ppm for CHG, with an S/N ratio surpassing 10.0. This validated method serves as a versatile tool for the accurate quantification of BKC, PCMX and CHG in cosmetics, hygiene products, health products and ophthalmic products. Its applicability extends across a wide range of industries, ensuring the reliable assessment of these critical biocides.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF EZETIMIBE IN RABBIT PLASMA USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objectives: The objective of the present work was to develop an analytical method and validation for the estimation of Ezetimibe in rabbit plasma using high-performance liquid chromatography (HPLC). Methods: A simple, rapid, sensitive, and accurate HPLC method was developed and validated for the quantification of Ezetimibe concentration in rabbit plasma using metoclopramide as an internal standard. Separation was performed on the Xerra C18 column (250 × 4.6 mm 5 μm) using a mobile phase consisting of 0.1% perchloric acid: acetonitrile(55:45 v/v) at a flow rate of 1 ml/min. Validation of the method was performed to demonstrate its selectivity, linearity, precision, accuracy, ruggedness, recovery, and matrix effect. Results: The calibration curves of Ezetimibe were linear over a concentration range of 5–1022 μg/mL. The with-in and between-day coefficients of variation were <10%. The extraction recoveries of Ezetimibe at the three levels of quality control samples were 99.961%, 99.767%, and 99.938%. Conclusion: The method was rapid with a retention time of Ezetimibe and the internal standard observed at 10 min, respectively. The developed method was successfully applied to studying the pharmacokinetics of Ezetimibe in rabbits.

Characterization of Forced Degradation Products of Netarsudil: Optimization and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Process-Related Impurities.

The aim of this study is to examine resolution, identification, and characterization of forced degradation products of netarsudil by liquid chromatography-tandem mass spectrometry by validating a simple and sensitive high-performance liquid chromatography method for the resolution, identification, and quantification of two process-related impurities in netarsudil. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB C18 (250 x 4.6 mm; 5 µ id) column at room temperature as the stationary phase and 257 nm as the detector wavelength with the mobile phase consisting of acetonitrile, methanol, and pH 4.6 phosphate buffer in 45:35:20 (v/v) at 1.0 mL/min flow rate in isocratic elution. The method reported very sensitive detection limits of 0.008 µg/mL for impurity 1 and 0.003 µg/mL for impurity 1. The method produces a calibration curve linear in the concentration level of 25-200 for netarsudil and 0.025-0.2 µg/mL for impurities. The proposed method gives acceptable results for other validation parameters such as accuracy, precision, ruggedness, and robustness. The drug was subjected to various stress conditions such as acid, base, peroxide, and thermal and ultraviolet light to investigate the stability-indicating ability of the method. Considerable degradation was observed in stress studies, and the degradation products were well resolved from process-related impurities. The characterization of degradation products was performed on the basis of collision-induced dissociation mass spectral data, and the possible structures of the six degradation compounds of netarsudil were proposed. The outcomes of other validation studies were likewise satisfactory and proven adequate for the regular analysis of netarsudil and its process-related impurities in bulk drug and pharmaceutical dosage forms and can also be applied for the evaluation of the stress degradation mechanism of netarsudil.

Stability Indicating HPLC Method for In-vitro Determination of Pantoprazole Sodium and its Degradation Products in Simulated Gastric and Intestinal Fluids

Background: A high-performance liquid chromatography (HPLC) method was developed for the determination of Pantoprazole Sodium (PPZ) in the presence of its degradation products. The degradation of PPZ was studied in simulated intestinal fluid (SIF) and simulated gastric fluids (SGF) in various temperature conditions. Aim: This study aimed to establish a simple, sensitive, and rapid RP HPLC method for in-vitro determination of Pantoprazole Sodium and its degradation products in simulated gastric and intestinal fluids. Objective: Pantoprazole is acid labile drug. In order to determine pantoprazole in various oral dosage forms, the stability-indicating assay of PPZ was performed in phosphate buffer (pH 6.8) representing simulated intestinal fluid (SIF) and in 0.1 molars (M) Hydrochloric acid (HCl) as simulated gastric fluid (SGF) at two different temperature conditions, i.e., 25°C and 0°C, respectively. Method: Pantoprazole sodium was obtained from the Akums laboratory in Haridwar. The analysis was performed by high-performance liquid chromatography (HPLC), Shimadzu, equipped with two LC-10 AD VP solvent-delivery modules, a SPD-10A UV–-visible detector, and a manual injector valve with 20 μL sample loop. Phenomenex ODS analytical column (150 mm × 4.6 mm i.d., 5 μm particles) was done under reversed-phase partition chromatographic conditions. The mobile phase was phosphate buffer and acetonitrile (ACN) of pH 7.4, respectively, optimized in a 70:30 (v/v) ratio followed by filtration through a 0.45 μm membrane filter and degassed by ultrasonicator before use. The mobile phase was delivered at the flow rate of 2 mL/min. The various parameters, such as linearity, accuracy and precision of the analytical method, were studied. Result: The standard curve of PPZ was linear (R2>0.99) over the concentration range of 5-30 μg/mL, and the relative standard deviation (RSD) values for intra-day and inter-day variations were in the range of 1.0-1.8%. The range of RSD was within ±2. Conclusion: The stability of PPZ in aqueous solution was pH dependent. The rate of degradation increases with decreasing pH. The pH stability of pantoprazole was studied at the above-mentioned temperature conditions. The PPZ peaks were analyzed by comparing them with fresh samples and were stable in SIF solution after 24 hours elapsed time at pH 6.8. The obtained degraded peaks in SGF (pH 1) were successfully separated from the PPZ.

Investigating a Simple and Sensitive High-performance Liquid Chromatography Method for Simultaneous Determination of Apigenin, Catalpol, and Gallic Acid Contents in Plantago Lanceolata L. and Plantago Major L.

Investigating a Simple and Sensitive High-performance Liquid Chromatography Method for Simultaneous Determination of Apigenin, Catalpol, and Gallic Acid Contents in Plantago Lanceolata L. and Plantago Major L.

Development and validation of an effective and sensitive technique for nitrate determination in fruits and vegetables using HPLC/PDA.

This study aims to develop an effective and sensitive HPLC (High Performance Liquid Chromatography) method to determine the nitrate concentration in fruits and vegetables (F & V) using a C18 column (ZORBAX Eclipse XDB-C18, 80Å, 250 × 4.6mm, 5μm (Agilent Technologies)) maintained at 40 0C, a mobile phase made up of methanol and buffer (pentane sulfonic acid sodium salt solution), and a Photo Diode Array Detector (PDA) at 225nm. The developed method is validated in terms of selectivity, linearity, accuracy, precision, suitability, the limit of detection (LOD), and the limit of quantification (LOQ) according to the European Union Decision 2002/657/EC. The result revealed that a ratio of 30: 70 of the organic modifier methanol and buffer with pH 2.8 shows the highest efficiency. The calibration curve shows linearity with a correlation coefficient (r) of 0.9985. The LOD and LOQ were found to be 2.26mg/kg and 7.46mg/kg. The recovery was in the range of 98.96-100.21%. Moreover, the greenness assessment scores of different approaches (eco-scale score of 76, AGREE score of 0.71, and few red shades in GAPI portray) were at a very excellent level. Thus, our developed method is fully validated and can determine the nitrate content in F & V.

Assessment on compatibility and safety of labels for pharmaceutical packaging

Although the secondary packing materials do not directly contact the finished drug products, compound migration may still happen between them. To ensure drug quality and safety, extractables and leachables of the packing materials should be analyzed. In this study, 2,6-di-tert-butyl-4-methylphenol (BHT) was first found in the labels for pharmaceutical packaging. For the identification of the compound, a strategy combining high performance liquid chromatography (HPLC), ultra-performance liquid chromatography-quadrupole time-of-flight mass (UPLC-Q-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy was utilized. Afterwards, a effective and sensitive HPLC method for quantification of BHT was developed and validated. Finally, a toxicological risk assessment of BHT was performed to ensure the safety of drugs.

Preparation, characterization, and pharmacokinetics of oridonin-loaded liposomes.

The aim of this study was to prepare oridonin liposomes and evaluate the physicochemical characteristics and pharmacokinetics in rats. A three-level, three-factor Box-Behnken design was used to optimize the preparation of oridonin liposomes. A highly sensitive high-performance liquid chromatographic quantification method using ultraviolet detection was established and validated for the determination of oridonin in rat plasma. Twelve Sprague-Dawley rats were randomly assigned and injected with 15 mg/kg of oridonin or oridonin liposomes via the tail vein. Pharmacokinetic parameters were estimated using a compartmental modeling approach using PKsolver software. The optimum conditions were as follows: soybean phospholipids/cholesterol ratio, 3.9:1; soybean phospholipids/drug ratio, 8.5:1; and soybean phospholipid concentration, 1.1%. Under these conditions, the mean particle size, polydispersity index, zeta potential, and encapsulation efficiency of oridonin liposomes were 170.5nm, 0.246, -30.3mV, and 76.15%, respectively. The pharmacokinetic results showed that liposomes could significantly prolong the elimination half-life (from 2.88 ± 0.55 to 13.67 ± 3.52 h), increase the area under the concentration-time curve (from 1.65 ± 0.17 to 6.22 ± 0.83 μg h/ml), and decrease the clearance (from 6.62 ± 1.38 to 1.96 ± 0.24L/kg h). The oridonin liposomes increased the elimination half-life and area under the concentration-time curve and provided a reference for the development of drugs with a short half-life.

Validation of a LC-UV method for determination of tildipirosin in goat milk

A simple, rapid, low-cost, and sensitive high-performance liquid chromatographic method was developed to determine tildipirosin in milk goat. Milk samples were precipitated with acetonitrile, and after evaporation, tildipirosin was determined by reverse-phase chromatography with an ultraviolet detector set at a wavelength of 289 nm. Tildipirosin was separated on a Zorbax Eclipse XDB-C18 column, 150 x 3.0 mm, 5 μm with gradient chromatographic elution. The retention times for tildipirosin and tylosin tartrate were 4.4 min and 10.5 min, respectively. Calibration curves were ranged from 100 to 2500 µg/L. The lower limit of detection was 75 µg/L, and the lower limit of quantitation was 100 µg/L. The accuracy and precision were always <15% except for LOQ < 20%. Mean recovery was 95.6 %. This procedure can be applied to perform pharmacokinetic studies in lactating animals and be useful to detect residues in dairy products.

Identification and quantification of extractables and leachables in laminated film and pouches for pharmaceutical packaging

Extractables and leachables from pharmaceutical packaging material can potentially be detrimental, which may affect the safety of the drug products. In this study, two compounds were found to be the possible extractables from the secondary packaging material. A strategy combining high performance liquid chromatography (HPLC), fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and nuclear magnetic resonance (NMR) spectroscopy was utilized for identification of the two compounds. Afterwards, a simple and sensitive HPLC method was developed and validated for the simultaneous quantification of both. The results indicated that this method was suitable for quantificating the two extractables in the pharmaceutical packaging material.

Chromatographic Analysis of Aqueous Humor of Bupivacaine in Different Administration Approaches.

Different administration approaches were investigated for the selection of bupivacaine administration type and a sensitive high-performance liquid chromatographic (HPLC) method has been developed. Developed method was validated and applied for the determination of bupivacaine in rabbit aqueous humor. The separation was achieved using a XTerra, C8 (250×8 mm i.d., particle size 5μm) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20mM; 30:70, v/v). Bupivacaine detection was performed by Diode Array detector (DAD) at 220 nm. The retention times for bupivacaine is 15.886 min. HPLC-DAD method was linear in the range of 75-4000 ng/mL. The limit of detection was 25 ng/mL and the limit of quantification of bupivacaine was found to be 75 ng/mL (relative standard deviation, RSD ≤ 15%, n = 6). In intra-day and inter-day precision and accuracy analysis, the RSD was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Method was carried out for three different type of bupivacaine application because of the investigation of effective drug administration. Twenty aqueous humor samples were in the range of 0.642 and 5.124 μg/mL.

ANALYTICAL METHOD VALIDATION OF GLICLAZIDE RELATED SUBSTANCES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD

Objective: The present study was undertaken with the objective of method validation of a rapid, simple, cost-effective HPLC method for the determination of related substances of Gliclazide. Methods: A simple, rapid, and specific method for analysis of gliclazide by a sensitive high-performance liquid chromatographic method is described. Validation of the method is carried out by USP and ICH guidelines. The method was validated for parameters like accuracy, precision, linearity, specificity, robustness, and system suitability. These proposed methods are suitable for the determination of title drugs in quality control laboratories in the pharmaceutical industries. Results: The mobile phase used for the chromatographic runs consisted of (450 ml) of acetonitrile and (550 ml) of water. The separation was achieved on the LiChroCART Supersher RP-8 column, (250 mm × 4.0 mm, 5 µm), using isocratic mode. Drug peaks were well separated and were detected by a UV detector at 235 nm, the method was linear at the concentration range. Gliclazide limit of detection (LOD) and limit of quantification (LOQ) was 0.003 and 0.01 while LOD and LOQ for Impurity-F were 0.003 and 0.01 respectively. Conclusion: The presented validated method is rapid, economic, simple, accurate, sensitive, robust, specific, and linear. It can be used for routine analysis of gliclazide.

Development and Validation of an Improved HPLC-UV Method for the Determination of Tildipirosin in Horse Plasma

Abstract A simple, rapid, low-cost, and sensitive high-performance liquid chromatographic method was developed to determine tildipirosin in horse plasma. Plasma samples were extracted with diethyl ether, and after evaporation, tildipirosin was determined by reverse-phase chromatography with an ultraviolet detector set at a wavelength of 289 nm. Tildipirosin was separated on a Zorbax Eclipse XDB-C18 column, 150 x 3.0 mm, 5 μm with gradient chromatographic elution. The retention times were 3.0 min and 6.4 min for tildipirosin and tylosin tartrate, respectively. The total run time was 9 minutes in this method. Calibration curves ranged from 0.1 to 3 μg/mL. The lower limit of detection for plasma was0.035μg/mL, and the lower limit of quantitation was 0.1 μg/mL. Both accuracy and precision were always < 12% exce pt for LLOQ < 20%. Mean recovery was 99.5 %. This procedure can be applied to determine tildipirosin concentrations in plasma and be useful to perform pharmacokinetic studies.

Development and validation of UV chromatographic method for quantification of copper and copper nanoparticles in different matrices and pharmaceutical products

Background The applications of Cu and CuNPs based on the earth-abundant and inexpensive Cu metal have generated a great deal of interest in recent years, including medical applications. A novel, specific, precise, accurate and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method with UV detection has been developed and validated to quantify copper (Cu) and copper nanoparticles (CuNPs) in different biological matrices and pharmaceutical products. Methods The developed method has been validated for linearity, precision, sensitivity, specificity and accuracy. Cu concentration was detected in pharmaceutical products without an extraction process. Moreover, liver, serum and muscle tissues were used as biological matrices. High Cu recovery in biological samples was afforded by using citric acid as a green chelating agent, exact extraction time and pH adjustment. Cu pharmaceutical and biological samples were eluted by acetonitrile: ammonium acetate (50 mM) with 0.5 mg/ml EDTA (30:70 v:v) as an isocratic mobile phase. EDTA reacted with Cu ions forming a Cu-EDTA coloured complex, separated through the C18 column and detected by UV at 310 nm. Results The developed method was specific with a short retention time of 4.95 min. It achieved high recovery from 100.3% to 109.9% in pharmaceutical samples and 96.8–105.7% in biological samples. The precision RSD percentage was less than two. The method was sensitive by achieving low detection limits (DL) and quantification limits (QL). Conclusion The validated method was efficient and economical for detecting Cu and CuNPs by readily available chemicals as EDTA and Citric acid with C18 column, which present the best results on RP-HPLC.

Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment).

Aspirin (also known as acetylsalicylic acid) is a drug intended to treat fever, pain, or inflammation. Treatment of moderate to severe cases of COVID-19 using aspirin along with dexamethasone has gained major attention globally in recent times. Thus, the purpose of this study was to use High-Performance Liquid Chromatography (HPLC) to evaluate the in vitro inhibition of CYP3A2 enzyme activity using aspirin in rat liver microsomes (RLMs). In this study, an efficient and sensitive HPLC method was developed using a reversed phase C18 column (X Bridge 4.6 mm × 150 mm, 3.5 µm) at 243 nm using acetonitrile and water (70:30 v/v). The linearity (r2 > 0.999), precision (<15%), accuracy and recovery (80–120%), limit of detection (5.60 µM and 0.06 µM), limit of quantification (16.98 µM and 0.19 µM), and stability of the newly developed method were validated for dexamethasone and 6β-hydroxydexamethasone, respectively, following International Conference on Harmonization (ICH) guidelines. This method was applied in vitro to measure CYP3A2 activity. The results showed that aspirin competitively inhibits 6β-hydroxylation (CYP3A2 activity) with an inhibition constant (Ki) = 95.46 µM and the concentration of the inhibitor causing 50% inhibition of original enzyme activity (IC50) = 190.92 µM. This indicated that there is a minimal risk of toxicity when dexamethasone and aspirin are co-administrated and a very low risk of toxicity and drug interaction with drugs that are a substrate for CYP3A2 in healthcare settings.

High-performance liquid chromatography quantitative analysis of ephedrine alkaloids in Ephedrae Herba on a perfluorooctyl stationary phase.

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R2 >0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87-110.99%), limits of detection (5.35-5.76µg/mL), and limits of quantification (20µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents ofthree batches of Ephedrae Herba from three pharmaceutical companies.

Analysis of cycloheximide in rat specimens using liquid chromatography with tandem mass spectrometry detection

The development of a selective and sensitive high-performance liquid chromatographic tandem mass spectrometric method for the determination of cycloheximide (CHX) in rat blood and plasma is described. The extraction of CHX and colchicine as internal standard from blood fluid (0.1 mL) was achieved using n-hexane: dichloromethane: isopropanol (20:10:1 v/v/v). The mobile phase, a combination of methanol:10 mM ammonium acetate (85:15, v/v), was pumped at 0.2 mL/min through a C18 analytical column with a run time of 3.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay exhibited excellent linearity (r2 > 0.999) in peak area response over the concentration ranges of 2–1000 ng CHX /mL blood fluid. The mean absolute recoveries for 20, 100 and 500 ng/mL CHX in blood fluid using the present extraction procedure were > 97%. The intra- and inter-day coefficients of variation in the plasma and blood and mean error were < 13% at different concentrations. Samples had limited stability at room temperature, and speedy processing is needed. After intravenous administration, rats had measurable concentrations of CHX for up to 24 h after dosing with 1 mg/kg of cycloheximide. The method displayed a high caliber of sensitivity and selectivity for detecting very low concentrations of CHX in rats.